Liver stem cells, or oval cells, proliferate in response to chronic liver injury and are believed to differentiate into both hepatocytes and cholangiocytes. We have recently identified a population of CD133+ murine oval cells that demonstrates bi-lineage (i.e. hepatocyte and cholangiocyte) gene expression at the single cell level. In this proposal, we will investigate oval cells phenotypes across multiple murine injury models. Our goal is to identify reliable oval cell immuno-phenotypes, so that we can further dissect the factors that influence proliferation and differentiation. Our hypothesis is that chronic liver injury results in growth factor stimulated proliferation and activates key factors in oval cell differentiation. We will utilize three complementary models of liver injury: 1) chronic oxidative stress (MAT1a KO), 2) environmentally-induced damage (DDC 0.1% diet), and 3) a tissue specific induced regulatory knockout (Pten liver KO). To test our hypothesis, we developed the following specific aims. Aim 1: Identify the oval cell phenotype in three models of chronic liver injury. Flow cytometry isolated CD133+ oval cells will be defined by bi-lineage potential and stem cell expression profile (Albumin, aFP, Ck19 and Hnf4a). Single cell in-vitro clonal analysis will document bi-potency. We will perform in-vivo functional analysis using bulk populations. Aim 2: Define the oval cell response to growth factors stimulation. Using quantitative PCR, we will demonstrate up-regulation of growth factor receptor expression. Using phospho-protein analysis, we will identify the intra-cellular phosphorylation events in growth factor stimulated oval cells. Lastly, we will define the response to growth factor receptor blockade in-vivo within oval cell population using cell proliferation assays. Aim 3: Dissect the role of transcription factor induced differentiation within populations of proliferating oval cells. We will downregulate HNF4a using siRNA to block differentiation in-vitro. We will isolate nuclear proteins from proliferating oval cells and control populations to define transcription factor profile important to oval cell differentiation. In terms of the relevance to public health, this research will provide the cornerstone of future developments to define the potential of adult stem cells in cellular based therapies for human liver disease. This proposal identifies the three key elements to oval cell based therapy: 1) reliable identification, 2) mechanisms of proliferation, and 3) key factors in differentiation.